Project Summary/Abstract Francisella tularensis is the causative agent of tularemia, the pneumonic form of which can be fatal even with appropriate treatment. This project builds on our recent identification (by contact PI Horwitz's group) of the Francisella Type VI Secretion System (T6SS) and on our subsequent determination (by PI Zhou's group) of the first atomic model of its sheath by cryo electron microscopy (cryoEM). T6SSs are large, complex and multi- protein nanomachines that Gram-negative bacteria use to sense environmental cues and deliver toxins into other bacteria or into eukaryotic hosts, as in the case of Francisella, where they mediate phagosome escape and intracytoplasmic replication. They are important virulence determinants, present in 25% of Gram-negative bacteria and in an even higher percentage of those that are human pathogens. However, our lack of knowledge concerning both their composition and structure has limited our understanding of their mechanisms of pathogenesis and our ability to ultimately design countermeasures against a myriad of bacterial diseases. The T6SS of Francisella is both significant and attractive to study because of the high infectivity and lethality of Francisella species and its relative simplicity compared with other T6SSs. However, significant knowledge gaps are yet to be filled including: (1) an atomic model of the structure of the pre-contraction outer sheath; (2) the composition and an atomic model of the baseplate and membrane core complex; and (3) the composition of the Francisella inner tube and secreted effector protein complex and an atomic model of its interaction with the sheath, baseplate, and membrane core complex in the pre-contraction state and during the contraction process. Towards this end, we propose to use the Francisella novicida T6SS as a model to advance our understanding of the T6SS, and verify our findings in the closely related F. tularensis live vaccine strain (LVS). We shall first obtain the atomic model of the sheath and tube complex in the purified T6SS in its pre- contraction state by single-particle cryoEM with electron-counting technology; then, by structure-guided mutagenesis and comparison with our atomic model of contracted sheath, we shall elucidate the energetics and mechanism of T6SS contraction. Second, by crosslinking, affinity pull-down, immunoblotting, proteomics, and bacterial 2-hybrid analyses, we shall determine the composition and protein interactions of the baseplate and membrane core complex. This information will be used in conjunction with cryoET of T6SS-containing minicells to determine the composition and structure of the T6SS baseplate and membrane core complex in their pre- and post-contraction states. Lastly, we shall determine the composition and structure of the Francisella T6SS effector (VgrG)-containing arrow complex. Elucidation of atomic level interactions among the protein components of the T6SS will guide further studies of its function and the development of new strategies for treating and preventing diseases caused by the numerous important pathogenic bacteria that have a T6SS.